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Hypochlorous Acid Staining with R19-S in the Drosophila Intestine upon Ingestion of Opportunistic Bacteria

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An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein

Authors: Yili Zhu, Weimin Tan and Wei-Lih Lee, date: 12/05/2018, view: 1165, Q&A: 0

[Abstract] In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by ...

FRET-based Stoichiometry Measurements of Protein Complexes in vitro

Authors: Francesca Mattiroli, Yajie Gu and Karolin Luger, date: 02/05/2018, view: 2704, Q&A: 0

[Abstract] For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a FRET-based assay to study protein complex stoichiometry in vitro. This assay, also known as Job plot, is set up as a continuous variation of the ...

Detection of Protein Interactions in the Cytoplasm and Periplasm of Escherichia coli by Förster Resonance Energy Transfer

Authors: Nils Y. Meiresonne, Svetlana Alexeeva, René van der Ploeg and Tanneke den Blaauwen, date: 01/20/2018, view: 3464, Q&A: 0

[Abstract] This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in Escherichia coli by Förster Resonance Energy Transfer (FRET). The described assay allows for the previously impossible in vivo screening of periplasmic protein-protein interactions. In FRET, excitation of a donor fluorescent ...

Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay

Authors: Ana Lechuga, Mónica Berjón-Otero, Margarita Salas and Modesto Redrejo-Rodríguez, date: 01/05/2018, view: 3309, Q&A: 0

[Abstract] This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules ...

Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions

Authors: Yohei Katoh, Kentaro Nakamura and Kazuhisa Nakayama, date: 01/05/2018, view: 3961, Q&A: 0

[Abstract] The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can ...

MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes

Authors: Maximilian G. Plach, Klaus Grasser and Thomas Schubert, date: 12/05/2017, view: 8096, Q&A: 0

[Abstract] Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides ...

Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells

Authors: Kaleeckal G. Harikumar, Yan Yan, Ting-Hai Xu, Karsten Melcher, H. Eric Xu and Laurence J. Miller, date: 11/20/2017, view: 7000, Q&A: 2

[Abstract] The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral overlap with the donor emission, is approximated with the donor. In the example provided, proteins ...

Protocol for Notch-ligand Binding Assays Using Dynabeads

Authors: Shogo Sawaguchi, Mitsutaka Ogawa and Tetsuya Okajima, date: 10/20/2017, view: 3495, Q&A: 0

[Abstract] This protocol describes how to measure interaction between Notch receptors and their ligands by cell-based assay using Dynabeads. We have used the protocol to determine binding capacity between Notch1-transfected HEK293T cells and ligand-coated Dynabeads. Expression of Eogt in Notch1-expressing cells promoted binding toward DLL4-coated ...

Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins

Authors: Xu Zheng and David Pincus, date: 06/20/2017, view: 3908, Q&A: 0

[Abstract] This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen peptide. Next, the bait protein is recaptured ...

Mating Based Split-ubiquitin Assay for Detection of Protein Interactions

Authors: Wijitra Horaruang and Ben Zhang, date: 05/05/2017, view: 6201, Q&A: 0

[Abstract] The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are ...

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